1. Field of the Invention
The present invention relates to a multiple sample container for reducing the time to detection (TTD) of biological activity in blood cultures and for mycobacteria in body fluids.
2. Background Description
The presence of biologically active agents such as bacteria in a patient's body fluid, especially blood, is generally determined using blood culture vials. A small quantity of blood is injected through an enclosing rubber septum into a sterile vial containing a culture medium, and the vial is then incubated at 37.degree. C. and monitored for microorganism growth.
One of the techniques used to detect the presence of microorganisms includes visual inspection. Generally, visual inspection involves monitoring the turbidity or eventual color changes of the liquid suspension of blood and culture medium. Known instrumental methods detect changes in the carbon dioxide content of the culture bottles, which is a metabolic by-product of the bacterial growth. Monitoring the carbon dioxide content can be accomplished by methods well established in the art, such as radiochemical or infrared absorption at a carbon dioxide spectral line. Until now, these methods have required invasive procedures which result in the well-known problem of cross-contamination between different vials. It has also been proposed to detect microorganism growth in sealable containers by monitoring positive and/or negative pressure changes.
Recently, non-invasive methods have been developed involving chemical sensors disposed inside the vial. These sensors respond to changes in the carbon dioxide concentration by changing their color or by changing their fluorescence intensity. In known automated non-invasive blood culture systems, individual light sources, spectral excitation/emission filters, and photodetectors are arranged adjacent to each vial. This results in station sensitivity variations from one vial to the next. Additional problems are caused by the aging effects of the light sources, filters and photodetectors. Due to the fact that most known blood culture sensors generate only a moderate contrast ratio in the measured photocurrent during bacterial growth, extensive and time-consuming calibration procedures and sophisticated detection algorithms are required to operate these systems. In addition, flexible electrical cables are required to connect the individual sources and detectors with the rest of the instrument. With the large number of light sources, typically 240 or more per instrument, maintenance can become very cumbersome and expensive when individual sources start to fail.
The disadvantage of intensity-based sensor arrangements can be overcome by utilizing fluorescent sensors that change their lifetime. In this case, intensity measurement is replaced with time parameter measurement, and intensity changes have no impact on the sensor output signal. Many chemical sensor materials are known that change their fluorescence lifetime with changing oxygen concentration, pH, carbon dioxide concentration, or other chemical parameters.
The change in the sensor fluorescence lifetime is commonly monitored by applying the well-known phase shift method. In this method, the excitation light is intensity-modulated, which results in an intensity-modulated fluorescence emission that is phase-shifted relative to the excitation phase. The phase shift angle, .theta., is dependent on the fluorescence lifetime .tau., according to the equation. EQU tan.theta.=.omega..tau. (1)
where .omega.=2.pi.f is the circular light modulation frequency.
An inspection of equation (1) reveals that the phase shift method allows for maximum resolution, d.theta./dr, under the condition .omega..tau.=1. Unfortunately, almost all known pH- or carbon dioxide-sensitive fluorophores have decay times in the range 5 ns to 500 ps. In other words, light modulation frequencies, f=1/2.pi..tau., in the range 32 MHz to 320 MHz would be required.
It is possible to accomplish light intensity modulation at such high frequencies, but that requires acousto-optic or electro-optic modulators that are only efficient in combination with lasers. Moreover, detecting the modulated fluorescence light would require high-speed high-sensitivity photodetectors such as microchannel-plate photomultipliers, which are rather expensive. Consequently, all commercial automated blood culture systems are based on intensity monitoring, and none of them utilize time-resolved fluorescent carbon dioxide sensors.
Even if it would be possible to operate fluorescence lifetime-based sensors at low cost, all sample-related artifacts would remain. In particular, the so-called "blood-background effect" has to be mentioned which is a continuous but unpredictable change in the fluorescence intensity and/or lifetime due to the metabolism of the blood itself. Because the blood-background effect can depend on the donor, the growth medium, the blood volume, and other factors, it is very difficult or impossible to distinguish between blood-related and organism-related fluorescence changes. Consequently, the detection algorithms have to be "robust" which results in a relatively long time to detection.